Growth hormone (GHRH) and pitutary adenylate cyclase activating peptide (PACAP) are the members of the PACAP/Glucagon superfamily, who are related in both sequence and function. Their stimulation of GH secretion and animal growth is concerned. A series of expression plasmid, pIRES1-GHRH-PACAP (P-G-P), pIRES1-GHRH (P-G) and pIRES1-PACAP(P-P), were constructed, extracted and purified, then transfected into CHO cell line with Lipofectamine. The expression was examined by RT-PCR, dot-ELISA and Western blotting. The biological activity of expression products was detected in rats. At 8 h after injection of transfection supernatant, serum IGF-Ⅰ concentrations in P-G-P group were significantly higher than that in other groups(P < 0.05). PLGA encapsulating plasmid microspheres were prepared and injected intramuscularly into rabbit legs. Growth behavior and IGF-1 level were measured at day 0, 15, 30 and 45 after injection. Greater body weights gain and higher serum IGF-Ⅰlevels were observed in three plasmid microsphere injection groups, compared with control group. At day 30, the body weight gain in P-G-P group was greater than saline group (81%, P < 0.01), P-G microsphere group (15%, P < 0.05) and P-P microsphere group (7%, P > 0.05), serum IGF-Ⅰ concentration in P-G-P microsphere group showed a 16.68% increase to P-G microsphere (P > 0.05), a 17.14% increase to P-P microsphere(P > 0.05) and a 50.46% increase to control (P < 0.05). These results suggest that co-expression of GHRH and PACAP in one expression plasmid might exert an additive stimulation of GH secretion and growth when delivered into rabbit skeletal muscle with PLGA microsphere. The results may provide a new approach to regulate animal growth.